Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

June 13, 1979

Pages 797-803

LIGHT-DEPENDENT PHOSPHORYLATION OF THYLAKOID MEMBRANE POLYPEPTIDES Richard

Bgliveau"

and Guy Bellemare#

DGpartement de Biochimie Facult6 des Sciences et de GEnie UniversitE Lava1 Qukbec, Qu6. GlK 7P4 Received

March

7 ,197 9

SUMMARY: The phosphorylation of thylakoid membrane proteins was studied using isolated chloroplasts from Euglena gracilis. We have found, using C3*P] labelling, that this phenomenon was light-driven, reversible in the dark, and completely inhibited by Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Polyacrylamide gel electrophoresis containing SDS has revealed five main bands which have been found to be proteins. Amino acid analysis of the bands has shown that ~3~P1 is incorporated into phosphothreonine. Many photochemical, in the

study

of photosynthetic

mational

change

Membrane

protein

Protein

kinase

from a cellular plasts it with

(3).

might

play

in the

activity fraction

coupling

has been

factor,

function

a role

in membrane

electron

flux,

electrical

reported

from Acetabularia

(2),

of this activation

study,

protein

using

isolated

transfer,

conforchange. activation.

chloroplasts

(1):

from pea chloro-

is

and coupling

reported

in this

and more recently, phosphorylation

have been

potential

seems to be a new phenomenon

The present membrane

events

activation: ion

of Brassica

The exact

thylakoid

and ultrastructural

membrane

phosphorylation

the ATP synthesis.

light-driven

molecular

still

unknown,

but

of photochemical chloroplasts

events shows a

phosphorylation.

MATERIALS AND METHODS Chloroplasts isolation: Euglena gracilis (strain 2) was grown photoheterotrophically, in the medium described by Price (4). Cells were harvested and washed twice with distilled water, followed by centrifugation at 1000 x g for 2 minutes, then washed again with grinding buffer (TB) modified from Salisbury KC1 replaced NaCl. The cells were resuspended in this buffer at 0.5 g/ml, is). broken in a French pressure cell at 1500 psi, and collected in 4 volumes Of grinding buffer. The broken cells were centrifuged at 100 x g for 1 minute in a small volume of TR buffer (4OmM Tricine-KOH pH 8.4; 0.33M Sorbitol), and *Fellow of the National Research Council of Canada # TO whom all correspondence should be sent.

and of Imperial

Oil

Ltd.

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by Academic Press, Inc. in any form reserved.

of reproduction

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMJNICATIONS

centrifuged successively at 100 x g for 5 minutes and then one minute, the pellet being discarded each time. Thylakoid membranes were isolated according to Vasconcelos (6). Isolated chloroplasts containing 50 ug of chloroIncorporation assays: phyll in 300 ul of TR, were incubated with carrier-free C32P!-orthophosphate (100 uC!i) from New England Nuclear, Boston. Incubation was at 20°C. The mixture was illuminated (10,000 lux) with two tungsten 15OW bulbs filtered to eliminate all wavelengths shorter than 580 nm. Aliquots of 50 ~1 were withdrawn at regular intervals, and plated on Whatman 3MM paper disks, which were treated according to Mans and Novelli (7). The filters were then counted by measuring Cerenkov radiation in 10 ml of water in a Beckman model LS 355 liquid scintillation spectrometer with a counting efficiency of 51%. Electrophoresis: The chloroplasts, after a 10 minute incubation, were centrifuged at 12000 x g for 10 set in a Eppendorf 3200 Microcentrifuge and immediately solubilized in a mixture of 5Om?4 Na2C03, 50mM dithiothreitol (DTT), 2% (wt/vol) SDS, 12% sucrose and 0.04% bromphenol blue, the final chlorophyll concentration of the preparation being 1 mg/ml. SDS gel electrophoresis was done according to Chua (8) using 7.5 to 15% polyacrylamide gels gradient; each slot contained 20 ul. Isolation of labelled amino acids: Regions of gels corresponding to the phosphorylated proteins were excised and trypsin-treated according to Clegg (9). After digestion and lyophilisation, the peptides were acid-hydrolised in 6N HCL The resulting amino acid mixture was again under N2, at 105'C for two hours. lyophilized, dissolved in water to remove trace of acid and lyophilized a third

0.1 10

20

TIME Fig.

1

a0

(ktd

Incorporation kinetics of [ 32P] orthophosphate into proteins by isolated Euglena chloroplasts. Incorporation in the light0 and incorporation in the light with CCCP 0 , incorporation in the dark 0 .

798

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

time, the amino acid mixture was then dissolved according chromatographed on Whatman 3MM paper, dried and put in a folder with one X Ray film RESULTS: first

The incorporation

10 minutes

10 minutes tion).

before

The control

in the

light,

then

of illumination, a slow

decrease

in the

in proteins

dark

it

of CCCP.

given

by the

radioautograph

among which

a plateau

(even with

six

(Fig.

are prominent

a

b

(10% v/v) and The paper was radioautography.

1) increases

shows no incorporation,

addition

bands,

(Fig.

reaches

in observed

but with

The pattern phorylated

of [x2Pl

in propan-2-01 to Jones (10). (RPR2X-Omat) for

for

during

an additional

continuous

illumina-

as does an other

2b) revealed

as seen in figure.

the

control

up to 10 phosBands I to

c

43000.

29000

I II

16890 14400

Fig.

2

proteins. In vitro incorporation of [32P1 orthophosp hate into chloroplast After electrophoresis, the gel is fixed with 50% methanol, 7% acetic acid and put in a folder with one X Ray film (RPR2X-Omat) for radioautographs . gel showing chloroplast proteins, a) stained b) radioautography showing chloroplast phosphoproteins, effect of proteinase K on sample. cl Proteinase K is used at 50 ug/ml, directly in the solubilisation mixture and incubation is clone for 2 hours at 37OC.

Vol. 88, No. 3, 1979

Fig.

3

BIOCHEMICAL

Location proteins

V are lost

of phosphoproteins. (C) and of isolated

after

treatment

RNase treatment treatment

total

peptides

of the

gels

and V (M,: as beeing

3).

12,900)

K, leaving

on any of the

membranes

Chromatographic permits

illumination

in the

proteinase

light

the

six

band

bands

(data

band VI (it give

establishing

phosphothreonine

When the one minute

thylakoid

RESEARCH COMMUNICATIONS

Radioautograph of total chloroplasts thylakoids membrane proteins (T).

at 90°C eliminates

chloroplasts, (Fig.

with

has no effect

The isolated the

AND BIOPHYSICAL

the

identification

nature

of labelled

(Fig.

is not

is

in the

800

shown).

dark

2~). TCA

of incorporation

as

of these

phosphorylated

bands

(M,:

II

of the phosphorylated

continuous,but

(Fig.

may be phospholipids).

and not phosphoserine

and one minute

not

same pattern

the membrane analyses

VI intact

23,500) amino

acids

4).

for (Fig.

alternating

periods

5),

incorporation

the

of

BIOCHEMKAL

Vol. 88, No. 3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

0

OJ 2

5

4

6

4

TINE

Fig.

4

Identification of C3*Pl sation of radioactivity PT phosphothreonine PS phosphoserine

Fig.

5

Effect of pulsed illumination on [ 32Pl incorporation into Conditions are as in Figure 1, but with 25 1.11 aliquots. alight off 0 bulk incorporation at the end of light period @bulk incorporation at the end of dark period

proteins.

the

to

follows is

therefore

brane, is

light under

during

represented

DISCUSSION: tides

is

stimulation.

The response Figure

one minute.

illumination,

and in the

by white

dots,

and the

As can be seen in Figure

light-dependent,

minutes.

labelled amino in threonine

The control

tion

at all,

the

thus

to be essential

the in the

coupling for

acid

time

shows the

dark.

The excited

ground

state,

1, the

phosphorylation

or in the

of photochemical phosphorylation

light events

to occur.

801

II

and V.

of the membrane

5 also

maximum incorporation dark,

in bands

two-states state,

by black

being with

Locali-

irradiation

of the in

mem-

illumination,

dots. of thylakoid

during

the

pep-

first

ten

CCCP, gave no incorpora-

to the ATP synthesis

seems

Vol. 88, No. 3, 1979

BIOCHEMICAL

The molecular 12,900)

is

quite

weights

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the two main phosphorylated

similar

to those

obtained

chloroplasts:

Mr:

26,000,

9,000,

more prominent

with

Euglena

chloroplasts,

are phosphorylated

instead

Our results

to the molecular

pattern

of incorporation,

major

phosphorylated

considering

the

Three

similarities

the

(3) , working

difference

that

and that

five

those

of Bennett

(th e amino

peptides, being

maximum

acid:

threonine.

divergences

possibilities

for

phosphate

incorporated

during

the

first

(I)

Fig.

6

shown not

with of these

weights

evolutionery

is

by Bennett

(Mr:

23,500,

with

band V is

well-defined

pea

definitly proteins

of two.

exhibit

regard

with

peptides

the the

fate

Illustration of three The beginning and end and OFF. I, II, III, ration continues (I), the dark period.

around

(during

case , since

dark

are our

not

reveal

minutes)

similarities

Eug,lena

the

period,

also

ten

These between

light

to be the

but

(3)

only

the

with

same

and the

same

are of interest,

and pea. period),

illustrated results

of the in Figure demonstrated

C32P1 6: no

possibilities in membrane protein phosphorylation. of illumination are indicated respectively by ON represent respectively situations where incorporemains constant (II) or decreases (III) during

802

Vol. 88, No. 3, 1979

BIOCHEMICAL

phosphorylation

in the

light-dependent

phosphorylation,

vation, tion

wh;le

third

of peptides,

We would between

thus two

observe,

(Fig.

the

peptides

dering

what

is

the

related

using

second

not

one would

pulse

give

decreases

related

to membrane

membrane

could

be chlorophyll-protein

known

about

membrane during

correct

activation. is

pulse

one.

iq stopped,

complex

coupling

of the

was the

acti-

photosynthesis.

incorporation

possibility

a

phosphoryla-

during

a transition

soon as irradiation

in photosynthetic

the

have revealed

activation

phosphate

the third

would

us a light-dependent

illimination, of [3*P!

5) shows that

(II)

necessarily

to membrane

The kinetics

implied

these

1); but

(III)

phosphorylation

is probably

(Fig.

possibly

states.

irradiation Since

the

dark

AND BIOPHYSICAL RESEARCH CCMMUNICATIONS

the

phenomenon

The ;ossibility of great

of photochemical

interest,

events

that consi-

to the ATP

synthesis. ACKNOWLEDGEMENTS: We wish to thank Johanne Saucier and G&ald Proteau for This work was supported by grants from National skillful1 technical assistance. Research Council No A6923 and FCAC (province de Q&bee). REFERENCES: 1.

Pai, H.S., Dehm, P., Schweiger, M., Rahmsdorf, H.J., Ponta, H., HirschKauffman, M. and Schweiger, H.G. (1975) Protoplasma 85, 209-218.

2.

Ralph,

R.K.,

McCombs,

P.J.A.,

Tener,

G. and Wojcik,

S.J.

(1972)

Biochem.

J. 130, 901-911. 3.

Bennet,

4.

Price,

5.

J.

(1977)

C.A.

Salisbury,

Nature

and Vallee, J.C.,

269, R.L.

Vasconcelos,

344-346. (1962) A.C.

Plant

Physiol.

and Floyd,

37,

G.L.

428-433.

(1975) Plant

Physiol.

56& 399-403. 6.

Vasconeelos, J.L. (1976)

A.C., Mendiola-Morgenthaler, Plant Physiol. s, 87-90.

7.

Mans,

R.J.

and Novelli,

G.D.

8.

Chua,

N.H.

and Bennoun,

P. (1975) P.N.A.S.

9.

Clegg,

.o .

Jones,

J.C.S.,

Brzeski,

(1961)

L.R.,

Arch.

H. and Kennedy,

Floyd,

Bioch.

Biophys.

(U.S.) '&

S.I.T.

G.L.,

(1976)

94,

Salisbury,

48-53.

2175-2179. J.

Gen. Virol.

413-430. J.

and Heathcote,

J.G.

(1966)

803

J.

Chromatog.

24,

106-111.

32,